It is known that bacteriolytic preparations are used as drugs in treatment for a number of diseases caused by pathogenic microflora, including that resistant to antibiotics.
The preparation having bacteriolytic effect—lysozyme is known (Egorov N. S. Bases of the doctrine of antibiotics, 1979, “Vysshaya shkola”, p. 347–349). Lysozyme of egg protein is active against a narrow spectrum of microorganisms, limited mainly by micrococci. By specificity of action on microbial cell wall peptidoglycan, lysozyme is a muramidase, i.e., lyzes the bond between muramic acid and glucosamine with the formation of fragments with muramic acid on the reducing end.
That is why lysozyme lyzes the narrow spectrum of gram-positive microorganisms. In particular, it does not lyze cell walls of staphylococci—gram-positive pathogenic bacteria, which are distinguished by the presence of pentaglycine interpeptide bridge that gives additional hardness to their cell wall. Besides, like most enzyme preparations, lyzozyme is not stable enough. All the above concerns also lysozymes from other, e.g., plant or bacterial, sources.
The enzyme preparation is also known, which is based on bacteriolytic enzymes—lysostaphin (Schindler C. A., Schuchardt V. T. Lysostaphin: a new bacteriolytic agent for the Staphylococcus. 1964, Proc. of the Nation, Acad. Sci. USA, v. 51, N 3, pp. 415–421).
However, it is unstable and applied in laboratory practice only.
The complex of bacteriolytic enzymes is known, which lyzes gram-positive microorganisms, in particular, pathogenic antibiotic-resistant staphylococci and other microorganisms, and which is obtained from culture liquid of bacterium Xanthomonas campestris VKPM B-4102 (patent RU 1549227, Int. class C12 N 9/00, 1984).
The complex of bacteriolytic enzymes from culture liquid of the bacterium Xanthomonas campestris is a basis of 2 drugs: the drug used in medicine was named lysoamidase and the drug used in veterinary—lysomast.
The drug consists of three bacteriolytic enzymes: muramidase, muramoylalanineamidase, endopeptidase, high-polymer polysaccharide, protease, and ballast components (O. A. Stepnaya, L. A. Ledova, I. S. Kulaev, Bacteriolytic enzymes, in: “Uspekhi biologicheskoy khimii”, v. XXXIX, Department of scientific and technical information, Pushchino Research Center of Russian Academy of Sciences, Pushchino, 1999, p. 341–346).
The ratio of components in the complex, determined by the authors, is (mas. %):
Bacteriolytic enzymes (muramidase, muramoylalanineamidase, endopeptidase)—1.0–2.0,
Protease—0.5–1.0,
Ballast components—4.0–8.0,
Polysaccharide—the rest.
The three bacteriolytic enzymes possess different substrate specificities to cell wall peptidoglycans of microorganisms: (a) muramidase is an analog of lyzozyme in specificity, (b) muramoylalanineamidase cleaves the peptide portion of peptidoglycan from the polysaccharide one, and (c) endopeptidase destroys the bonds within peptide bridges of the cell wall. Due to the above, the drug actively lyzes cells of a wide range of gram-positive microorganisms, including pathogenic streptococci and staphylococci.
Due to the presence of protease and two of the bacteriolytic enzymes of lysoamidase—endopeptidase and muramoylalanineamidase that possess also proteolytic activity, the drug is good for cleaning wounds from necrotic masses.
The polysaccharide constituent of the complex provides stability of the bacteriolytic enzymes (O. A. Stepnaya, L. A. Ledova, I. S. Kulaev, Biochemistry [in Russian], v. 58, 10, 1993, p. 1523–1528). The polysaccharide structure is formed by a repeating unit, consisting of N-acetylglucosamine, N-acetylmannuric and N-acetylgalacturonic acids (L. M. Likhosherstov, S. N. Senchenkova, Y. A. Knirel, et al. FEBS Letters, N 368, 1995, p. 113–116).
The disadvantage of this complex is that its bacteriolytic activity is not high enough due to the presence of ballast components and because nearly the half of the bacteriolytic enzymes are denatured. Besides, the ballast components have no medicinal effect but may cause allergic reactions.
The method of production of a complex of lytic enzymes is known (patent RU 1774658, Int. class C12N 9/36, 1990), which includes cultivation of the strain-producer Xanthomonas campestris VKPM B-4102 in a fluid nutrient medium containing glucose, bactopeptone, protein-vitamin concentrate (PVC) in the amount of 2.5–6.0 g/l, and mineral salts, under aeration and stirring, followed by isolation of the target product, with some part of PVC being added to the initial medium. The content of bacteriolytic enzymes in the culture liquid is 40–60 U/ml in bacteriolytic activity units.
Isolation is realized by the method of isolation of the lytic enzyme complex described in the patent RU 1755581 (Int. class C12 N 9/36, 1990), which includes the cooling of culture liquid filtrate, precipitation of the target product by an organic solvent, cooling and separation of precipitate, dialysis and lyophilization. Before precipitation, the filtrate is cooled, acidified to pH 3.5–5.0 followed by addition of 2.5–3.5 v of cooled ethanol, and held in cold place without stirring.
The content of bacteriolytic enzymes in the preparation is no more than 50 U/mg preparation in bacteriolytic activity units.
The yield of finished product is 40–60% of the content of the lytic complex in culture liquid.
Both the content of bacteriolytic enzymes in culture liquid and the yield of finished preparation are not high enough at realization of this method.
The closest to the method proposed, as regards the totality of significant attributes and the effect achieved, is the method of production of a lytic enzyme complex (patent RU 1549227, Int. class C12 N 9/00, 1984) that provides for cultivation of the bacterium Xanthomonas campestris VKPM B-4102 on a nutrient medium with the following components (g/l):
Glucose 2.0–12.0Bactopeptone 2.0–6.0Protein-vitamin concentrate 2.5–6.0Na2HPO4 × 12H2O 0.5–4.0KH2PO4 0.1–1.0NaCl0.35–1.0MgSO4 × 7H2O 0.2–3.0FeSO4 × 7H2O0.01–0.1Waterup to 11
The process is stopped when a stable lytic activity of culture liquid is reached. Then the biomass is separated by centrifugation, the complex of the lytic enzymes present in the solution is isolated by precipitation with ammonium sulfate, the precipitate is dissolved, and the solution is dialyzed and lyophilized.
The content of bacteriolytic enzymes in culture liquid is no more than 63 U/ml in bacteriolytic activity units.
The yield of end product is not high enough—about 20% of the lytic complex content in culture liquid.
The above method has a disadvantage that the protein-vitamin concentrate is currently not produced by domestic industry, because its production is ecologically unsafe; the use of expensive imported bactopeptone Difco (USA) as a medium component is unpractical, because it significantly increases the cost of the final product.
Besides, the use of ethanol (RU 1755581) and acetone (RU 1549227) for precipitation of the enzymes from culture liquid of the producer results in irreversible denaturing of about the half of bacteriolytic enzymes determining the therapeutic effect of the drug, which are present in the culture liquid (according to the results of bacteriolytic activity measurement and electrophoretic analysis) and, consequently, in a considerable decrease in the product yield (FIG. 1).
Finished lysoamidase preparations produced by the known methods contain a significant amount of minor components—ballast proteins (FIG. 1), which have no medicinal effect but may cause an allergic reaction.
The strain-producer of the complex is known: bacterium Xanthomonas campestris VKPM B-4102.
The disadvantage of this strain-producer is that, due to the presence of minor components in the produced complex, bacteriolytic activity is insufficiently high.